The present invention relates to a process for the microbial production of (4R,6R)-4-hydroxy-2,2,6-trimethylcyclohexanone (xe2x80x9cactinolxe2x80x9d) from (6R)-2,2,6-trimethylcyclohexanedione (xe2x80x9clevodionexe2x80x9d). Actinol is useful for the synthesis of carotenoids, such as zeaxanthin. More particularly, the present invention relates to a process for the microbial production of actinol utilizing a specific microorganism that is capable of selectively asymmetrically reducing the carbonyl group at the C-4 position of levodione.
Actinol has previously been prepared by optical resolution of the diastereomeric mixture of actinol. However, this process requires hydrogenation of levodione by metal catalysts and subsequent optical resolution by chemical means with resolving agents, such as, maleic anhydride (T. Ohashi et al., the proceedings of the symposium xe2x80x9cMolecular Chirality 1996xe2x80x9d held on May 30 and 31, 1996, in Tokyo, Japan, pages 47 to 50, xe2x80x9cPractical Syntheses using Biocatalystsxe2x80x9d). Accordingly, this process is not economically feasible for industrial purposes.
Processes of enzymatic preparation of actinol from levodione per se are known. For example, Bacillus thermophilus is capable of converting racemic dihydrooxoisophorone to 4 isomers of 4-hydroxy-2,2,6-trimethylcyclohexanone, i.e., to the cis-(4R,6S)-, cis-(4S,6R)-, trans-(4R,6R)- and trans-(4S,6S)-isomers. The resulting quantitative ratio of these isomers is 68:25:5:2 (J. Biotechnol., 9(2), 117 -128, 1989). Because the content of the (4R,6R)-isomer, actinol, is only 5% of the total isomers, this process is also not economically feasible for industrial purposes.
As a result of extensive studies on selective asymmetric reduction of levodione, it has surprisingly been found that actinol can be obtained efficiently from levodione by selective asymmetric reduction using certain microorganisms followed by recovery of the actinol from the reaction mixture. The present invention is based upon this finding.
Accordingly, the present invention provides a process for making actinol by contacting levodione with a microorganism, which is selected from the group consisting of microorganisms of the genera Cellulomonas, Corynebacterium, Planococcus and Arthrobacter and which is capable of selective asymmetric reduction of levodione to actinol, and recovering the resulting actinol from the reaction mixture.
Screening was effected using a method known per se. For example, a microorganism is cultivated in a nutrient medium containing saccharides, such as, glucose and sucrose, alcohols, such as, ethanol and glycerol, fatty acids, such as, oleic acid and stearic acid or esters thereof, or oils, such as, rapeseed oil and soybean oil as carbon sources; ammonium sulfate, sodium nitrate, peptone, amino acids, corn steep liquor, bran, yeast extract and the like as nitrogen sources; magnesium sulfate, sodium chloride, calcium carbonate, potassium monohydrogen phosphate, potassium dihydrogen phosphate and the like as inorganic salt sources; and malt extract, meat extract and the like as other nutrient sources by a conventional method to provide cells. The cultivation can be carried out aerobically, normally for a cultivation period of 1 to 7 days at a medium pH of 3 to 9 and a cultivation temperature of 10 to 40xc2x0 C. After the cultivation, the resulting cells are collected by centrifugation or filtration. The cells thus obtained and levodione are brought (contacted) together in a solvent such as water, potassium phosphate buffer, acetonitrile, ethanol and the like, and a reaction is initiated under appropriate reaction conditions (levodion concentration: 400 to 2000 mg/g dry cells/l, pH range: 4 to 9, temperature range: 20 to 50xc2x0 C., reaction period: 10 minutes to 80 hours). The reaction mixture is extracted with an organic solvent such as ethyl acetate, n-hexane, toluene, n-butyl acetate and the like. The extracted solution is subjected to an appropriate method, such as, chromatography, to measure the productivity of actinol from levodione.
As a result of the screening, it has been found that microorganisms belonging to the genera Cellulomonas, Corynebacterium, Planococcus and Arthrobacter are capable of the selective asymmetric reduction of levodione. Preferred such microorganisms are Cellulomonas sp. AKU672, Corynebacterium aquaticum AKU610, Corynebacterium aquaticum AKU611, Planococcus okeanokoites AKU152 and Arthrobacter sulfurous AKU635. Especially preferred are the first three named microorganisms, of which Corynebacterium aquaticum AKU611 is most preferred.
The microorganisms Cellulomonas sp. AKU672, Corynebacterium aquaticum AKU610 and Corynebacterium aquaticum AKU611 were isolated from soil samples collected at Lake Manahime, Fukui Prefecture, Japan. These micoorganisms were deposited with the National Institute of Bioscience and Human-Technology, Agency of Industrial Science and Technology, Japan on Aug. 4, 1998 under the Budapest Treaty and have the following designations: Cellulomonas sp. AKU672 (FERM BP-6449) Corynebacterium aquaticum AKU610 (FERM BP-6447) Corynebacterium aquaticum AKU611 (FERM BP-6448)
Cellulomonas sp. AKU672, Corynebacterium aquaticum AKU610, and Corynebacterium aquaticum AKU611 were deposited on Aug. 4, 1998 at the National Institute of Bioscience and Human-Technology, Agency of Industrial Science and Technology (1-3, Higashi 1-Chome, Tsukuba City, Ibaraki Pref., Japan) as FERM BP-6449, FERM BP-6447, FERM BP-6448, respectively under the terms of the Budapest Treaty. All restrictions on public access to these deposits will be irrevocably removed upon the grant of a patent on this application.
These three microorganisms, and also Planococcus okeanokoites AKU152 and Arthrobacter sulfureus AKU635, are new and represent a further aspect of the present invention.
The above-mentioned strain AKU672 (FERM BP-6449) has the following taxonomical properties:
Typical pleomorphism of strain Cellulomonas sp. AKU672 was found on electron microscopic observation. An old culture of the strain was coccoidal as shown in FIG. 1. In young cultures, irregular rods were dominant (FIG. 2). The morphological, physiological and biochemical characteristics of the strain are summarized in Tables I and II.
The strain Cellulomonas sp. AKU672 is gram-positive and aerobic, and can be classified as belonging to the group of xe2x80x9ccoryneform bacteriaxe2x80x9d. This strain was motile with one flagellum. Ornithine was found in the cell wall as the principal amino acid. Its content according to gas chromatography (xe2x80x9cGCxe2x80x9d) was found to be 74.7%. Bending-like cell division was observed. The strain produced acid from a wide variety of sugars without gas formation for 4 days. This strain did not show cellulolytic activity.
The classification of coryneform bacteria is not well established. Recently, Yamada and Komagata [J. Gen. Appl. Microbiol., 18, 417 (1992)] proposed classifying the coryneform bacteria into seven groups depending on the principal type of cell division, cell wall composition and DNA content according to GC. They differentiated Group 4 from other groups despite the lack of cellulolytic activity. Bacteria of this group exhibit the bending type of cell division, and the principal amino acid in the cell wall is ornithine. Their contents according to GC are distributed in a narrow and high range from 71 to 73%. These bacteria produce acid fermentatively from a wide variety of sugars. According to their proposal, the strain Cellulomonas sp. AKU672, which did not show cellulolytic activity, should belong to Group 4. Other characteristics of the strain on classification coincided well with those of Group 4, and so it has been tentatively named as Cellulomonas sp. AKU672.
The above-mentioned strains AKU610 and AKU611 have the following taxonomical properties:
1) Growable temperature: 15-40xc2x0 C.
2) Optimum temperature for growth: 30xc2x0 C.
3) Obligatory aerobic and gram negative microorganism
4) Spore formation: None
5) Polymorphism and traditional rod-cocus cycles can be observed during cultivation.
6) Motility: None
Moreover, the strains Corynebacterium aquaticum AKU610 and AKU611 were identified as such based on assimilation of various carbon sources by the Biolog System (Biolog Inc., 3447 Investment Blvd., Suite 3, Hayward, Calif. 94545, USA: Nature Vol. 339, 157-158, May 11, 1989) as follows:
Cells of each strain were inoculated with 96-well microtiter-plates and incubated for 24 hours at 28xc2x0 C. Each well contains one of 96 kinds of carbon sources in BUGM+B medium (Biolog Universal Growth Media+blood; Biolog Inc.).
After incubation, each strain showed the following assimilation of carbon sources:
From the above results, both strains are identified as Corynebacterium aquaticum and named Corynebacterium aquaticum AKU610 and AKU611, respectively.
Other microorganisms mentioned above are available from a public depositary (culture collection) to anyone upon request, such as the Institute of Fermentation Osaka, Japan (IFO). Examples of such deposited strains are Planococcus okeanokoites AKU152 (IFO 15880) and Arthrobacter sulfureus AKU635 (IFO 12678).
The selective asymmetric reduction process of the present invention can be carried out batchwise, semibatchwise or continuously in water or in a solvent medium that is miscible with water, enhances levodione solubility and is inert to the enzyme reaction, such as, 0.01 to 0.5M potassium phosphate buffer, another buffer with the pH range 4 to 10, acetonitrile, ethanol or N,N-dimethylformamide. The concentration of levodione is conveniently 400 to 2000 mg/1 g dry cells/l, preferably 400 to 800 mg/1 g dry cells/l. The selective asymmetric reduction process may be carried out in a pH range from 4 to 9, preferably from 6 to 7, in a temperature range from 20 to 50xc2x0 C., preferably 30 to 40xc2x0 C., and for 10 minutes to 80 hours, preferably for 8 hours to 24 hours.
The selective asymmetric reduction process of the present invention is conveniently carried out in the presence of a co-factor such as nicotinamide adenine dinucleotide (NAD), nicotinamide adenine dinucleotide phosphate (NADP), or said co-factor with glucose and glucose dehydrogenase (GDH). The concentration of such co-factor in the reaction medium is preferably 300 mM/l or more, more preferably from 700 mM/l to 900 mM/l. Moreover, the yield of actinol can be increased by addition of a surfactant to the reaction mixture. Span(copyright) 20, Span(copyright) 80, Tween(copyright) 20, Tween(copyright) 40 (all available from Wako Pure Chemical Ind., 3-1-2 Dosho-machi, Osaka, Japan) and the like are examples of surfactants that can be used. The amount of surfactant in the reaction medium is conveniently 2 to 20 mM/l, preferably about 8 mM/l.
After selective asymmetric reduction has been completed, the actinol thus obtained can be recovered by extraction with a water-insoluble (water- immiscible) organic solvent that readily solubilizes actinol, such as, ethyl acetate, n-hexane, toluene or n-butyl acetate. Further purification of actinol can be effected by concentrating the extract to directly crystallize the actinol or by the combination of various kinds of chromatography, such as, thin layer chromatography, adsorption chromatography, ion-exchange chromatography and/or gel filtration chromatography. If necessary, high performance liquid chromatography can also be applied. A preferred recovery leading to crystals of actinol involves extracting the actinol with ethyl acetate and concentrating the extract to obtain actinol crystals.
As an alternative to the above described xe2x80x9cresting cell reactionxe2x80x9d technique, actinol can be produced by fermentation of the above microorganisms in a nutrient medium in the presence of levodione, i.e., in a xe2x80x9cgrowing cell reactionxe2x80x9d. Both alternatives are embraced by the process of the present invention.
As nutrient media in the xe2x80x9cgrowing cell reactionxe2x80x9d technique there may be used those which contain saccharides, such as, glucose and sucrose, alcohols, such as, ethanol and glycerol, fatty acids, such as, oleic acid and stearic acid or esters thereof, or oils, such as, rapeseed oil and soybean oil as carbon sources; ammonium sulfate, sodium nitrate, peptone, amino acids, corn steep liquor, bran, yeast extract and the like as nitrogen sources; magnesium sulfate, sodium chloride, calcium carbonate, potassium monohydrogen phosphate, potassium dihydrogen phosphate and the like as inorganic salts; and malt extract, meat extract and the like as other nutrient sources. As a further aspect of the present invention, actinol can be produced by fermentation of the above microorganisms in a nutrient medium in the presence of levodione.
The fermentation can be carried out aerobically, normally for an incubation period of 1 to 7 days at a medium pH of 3 to 9 and a fermentation temperature of 10 to 40xc2x0 C.
The microorganisms to be used in the fermentation may be in any form, for example, cultures obtained by fermentation of strains in liquid media, cells separated from liquid cultures, dried cells obtained by processing cells or cultures, or immobilized cells.